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Chemiluminescent reagent

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Chemiluminescent reagent is a laboratory product designed to produce light through a chemical reaction. It is commonly used in various analytical techniques, such as immunoassays and Western blotting, to detect and quantify specific target molecules.

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Lab products found in correlation

Las 3000 imaging system, by Fujifilm (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Protease inhibitor cocktail, by Merck Group (3 mentions) Protean ief cell, by Bio-Rad (2 mentions) λ phosphatase, by New England Biolabs (2 mentions) Ipg strips with 4 7ph range, by Bio-Rad (2 mentions) Phos tag page, by Fujifilm (2 mentions) Antibody stripping solution, by Merck Group (2 mentions) Coomassie brilliant blue, by Bio-Rad (2 mentions) Anti β actin monoclonal antibody, by Merck Group (1 mentions) Chip it express kit, by Active Motif (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Acetylated histone h4, by Merck Group (1 mentions) Phosphorylated jnk, by Cell Signaling Technology (1 mentions) M35 a x omat processor, by Kodak (1 mentions) P jnk, by Cell Signaling Technology (1 mentions) Total jnk, by Cell Signaling Technology (1 mentions) Degs 1, by Novus Biologicals (1 mentions) Imagequant las 4000 system, by GE Healthcare (1 mentions)

Spelling variants of this product

Enhanced chemiluminescent reagent (11 citations) Western Lightning chemiluminescent reagent (3 citations) ECL chemiluminescent detection reagent (3 citations) Western Lighting Plus-ECL Chemiluminescent Reagents (2 citations)

12 protocols using chemiluminescent reagent

1

Protein Phosphorylation Analysis Protocol

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Total cell lysates were prepared in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% NaDoc, 0.1% SDS, 50 mM Tris pH 8) containing protease and phosphatase inhibitors. Protein concentration was measured using bovine serum albumin as a standard58 (link). Isoelectric focusing (IEF) was done using the Protean IEF Cell (Bio-Rad) at 20°C with rapid ramping to voltage 10,000 V at a current limit of 50 μA using IPG Strips with 4–7pH range (Bio-Rad). Where indicated total cellular fractions were treated with λ-phosphatase according to manufacturer’s instructions (New England BioLabs) before isoelectric focusing. Phos-tag PAGE was performed according to manufacturer’s instructions (Wako) using 100 μM Phos-tag. Samples were subjected to SDS-PAGE, transferred to nitrocellulose membrane, blocked with low-fat milk and incubated with primary antibody overnight. The proteins were visualized by using peroxidase-conjugated secondary antibodies and chemiluminescent reagent (PerkinElmer) in LAS-3000 Imaging System (Fujifilm). Densitometric quantification was performed on unsaturated images using ImageJ.
Kaushik S, & Cuervo A.M. (2015). Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis. Nature cell biology, 17(6), 759-770.
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2

Western Blot Analysis of Cellular Proteins

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Cells were lysed in a lysis buffer containing Tris-EDTA, 1% SDS, 1 mM DTT, 2 mM sodium vanadate, and protease inhibitor cocktails (Sigma-Aldrich). Cytosolic and nuclear proteins were extracted using the Pierce kit (Pierce, Rockford, IL). Proteins (20–50 μg) were loaded on freshly prepared SDS-PAGE gels. Resolved proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA) and probed by antibodies. Membranes were exposed to chemiluminescent reagent (PerkinElmer, Waltham, MA) and visualized on Kodak films. In all experiments, after being probed by antibodies for target proteins, PVDF membranes were stripped with antibody-stripping solution from EMD Millipore (Billerica, MA) and reimmunoblotted with antibodies for internal controls such as β-actin. Results of Western blots were quantified by AlphaEaseFC.
YANG C, & JIANG Q. (2018). Vitamin E δ-tocotrienol inhibits TNF-α-stimulated NF-κB activation by up-regulation of anti-inflammatory A20 via modulation of sphingolipid including elevation of intracellular dihydroceramides. The Journal of nutritional biochemistry, 64, 101-109.
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3

Investigating Glucocorticoid Receptor Activation

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LPS, DEX, 1,25(OH)2D3 (VitD), an active form of vitamin D, and monoclonal anti-β-actin antibody were purchased from Sigma (St. Louis, MO). HyQTase was purchased from Hyclone Laboratories (Logan, UT). Rabbit polyclonal antibody to GR and Rabbit IgG were purchased from Abcam, Inc. (Cambridge, MA). Rabbit polyclonal antibody to GR phosphorylated at S211 was purchased from Cell Signaling (Danvers, MA). Rabbit polyclonal antibody to histone H4 and acetylated histone H4 were purchased from Millipore (Temecula, CA). Anti-mouse and anti-rabbit horseradish peroxidase (HRP)-labeled IgG were purchased from Amersham Biosciences (Piscataway, NJ). Chemiluminescent reagent was purchased from Perkin Elmer Life Sciences (Waltham, MA). ChIP-IT Express kit was purchased from Active Motif (Carlsbad, CA). Mouse monoclonal antibody to GR and all the antibodies and reagents used for flow cytometry were purchased from BD Biosciences (San Diego, CA).
Zhang Y., Leung D.Y, & Goleva E. (2014). Anti-inflammatory and Corticosteroid Enhancing Actions of Vitamin D in the Monocytes of Steroid Resistant and Steroid Sensitive Asthmatics. The Journal of allergy and clinical immunology, 133(6), 1744-1752.e1.
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4

Protein Phosphorylation Analysis Protocol

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Total cell lysates were prepared in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% NaDoc, 0.1% SDS, 50 mM Tris pH 8) containing protease and phosphatase inhibitors. Protein concentration was measured using bovine serum albumin as a standard58 (link). Isoelectric focusing (IEF) was done using the Protean IEF Cell (Bio-Rad) at 20°C with rapid ramping to voltage 10,000 V at a current limit of 50 μA using IPG Strips with 4–7pH range (Bio-Rad). Where indicated total cellular fractions were treated with λ-phosphatase according to manufacturer’s instructions (New England BioLabs) before isoelectric focusing. Phos-tag PAGE was performed according to manufacturer’s instructions (Wako) using 100 μM Phos-tag. Samples were subjected to SDS-PAGE, transferred to nitrocellulose membrane, blocked with low-fat milk and incubated with primary antibody overnight. The proteins were visualized by using peroxidase-conjugated secondary antibodies and chemiluminescent reagent (PerkinElmer) in LAS-3000 Imaging System (Fujifilm). Densitometric quantification was performed on unsaturated images using ImageJ.
Kaushik S, & Cuervo A.M. (2015). Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis. Nature cell biology, 17(6), 759-770.
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5

Immunoblot Analysis for Protein Quantification

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Immunoblot analysis was performed using the indicated primary antibodies and horseradish peroxidase-conjugated anti-mouse IgG (NA931-1ML), anti-rabbit IgG (NA934-1ML; GE Healthcare), or anti-goat IgG (6420-05; SouthernBiotech, Birmingham, AL) as secondary antibodies. Upper and lower portions of the immunoblot membranes were cut based on molecular mass markers prior to incubation with indicated primary antibodies. A chemiluminescent reagent (PerkinElmer Life Sciences, Waltham, MA) was used for signal detection of immunoblots, followed by quantification of immunoreactivity using the ImageJ software38 (link). Protein concentrations in the samples were estimated using Coomassie Brilliant Blue (Bio-Rad Laboratories, Inc.), with BSA as a standard. Student’s t-tests (two-tailed) were used to evaluate the statistical significance of two-group comparisons. Statistical significance was set at p < 0.05.
Yoshida A., Ohtsuka S., Matsumoto F., Miyagawa T., Okino R., Ikeda Y., Tada N., Gotoh A., Magari M., Hatano N., Morishita R., Satoh A., Sunatsuki Y., Nilsson U.J., Ishikawa T, & Tokumitsu H. (2024). Development of a novel AAK1 inhibitor via Kinobeads-based screening. Scientific Reports, 14, 6723.
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6

Western Blot Protein Analysis

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Cells were lysed in a lysis buffer containing Tris-EDTA, 1% SDS, 1 mM DTT, 2 mM sodium vanadate and protease inhibitor cocktails (Sigma). Cytosolic and nuclear protein were extracted using the Pierce Kit (Pierce, Rockford, IL). Proteins (20–50 μg) were loaded on BioRad pre-cast SDS-PAGE gels. Resolved proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA) and probed by antibodies. Membranes were exposed to chemiluminescent reagent (Perkin Elmer, Waltham, WA) and visualized on a Kodak film. In all the experiments, after probed by antibodies for target proteins, PVDF membranes were stripped with antibody-stripping solution from EMD Millipore Corporation (Billerica, MA) and re-immunoblotted with antibodies for internal controls such as β-actin. Results of Western blots were quantified by ImageJ.
WANG Y., PARK N.Y., JANG Y., MA A, & JIANG Q. (2015). Vitamin E γ-tocotrienol inhibits cytokine-stimulated NF-κB activation by induction of anti-inflammatory A20 via stress adaptive response due to modulation of sphingolipids. Journal of immunology (Baltimore, Md. : 1950), 195(1), 126-133.
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7

Protein Quantification and Western Blot Analysis

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Total cell lysates were prepared in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% NaDoc, 0.1% SDS, and 50 mM Tris, pH 8) containing protease and phosphatase inhibitors. Protein concentration was measured using BSA as a standard (Lowry et al., 1951 (link)). Samples were subjected to SDS-PAGE, transferred to nitrocellulose membrane, blocked with low-fat milk, and incubated with primary antibody overnight. The proteins were visualized by using peroxidase-conjugated secondary antibodies and chemiluminescent reagent (PerkinElmer) in a LAS-3000 Imaging System (Fujifilm). Densitometric quantification was performed on unsaturated images using ImageJ.
Gong Z., Tasset I., Diaz A., Anguiano J., Tas E., Cui L., Kuliawat R., Liu H., Kühn B., Cuervo A.M, & Muzumdar R. (2018). Humanin is an endogenous activator of chaperone-mediated autophagy. The Journal of Cell Biology, 217(2), 635-647.
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8

Western Blot Analysis of Cellular Proteins

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Cells were lysed in lysis buffer containing Tris-EDTA, 1% SDS, 2 mM Na3VO4 and protease inhibitor cocktails (Sigma). Equal amount of proteins (15–30 μg) were separated on acrylamide gels by SDS-electrophoresis and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bilerica, MA, USA), and probed by antibodies. Membranes were exposed to chemiluminescent reagent (PerkinElmer) and visualized on Kodak film with an M35A XOmat processor (Kodak). The antibodies used in our study were as follows: DEGS1 (Novus Biologicals, Littleton, CO), membrane bound microtubule-associated protein light chain 3 (LC3; MBL international, Woburn, MA), poly (ADP-ribose) polymerase-1 (PARP-1), HMG-CoA reductase, actin (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated JNK at Thr-183 and Tyr-185 (p-JNK), and total JNK (Cell Signaling Technology, Danvers, MA).
JANG Y., RAO X, & JIANG Q. (2017). Gamma-tocotrienol profoundly alters sphingolipids in cancer cells by inhibition of dihydroceramide desaturase and possibly activation of sphingolipid hydrolysis during prolonged treatment. The Journal of nutritional biochemistry, 46, 49-56.
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9

Purification and Detection of Protein Complexes

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Soluble PDGFRα fused to an Fc domain and HA epitope tag was expressed in 2936E cells and coupled to protein A agarose as previously described [33 (link)]. Purified soluble trimer complexes were added to 1ml of Tris-saline buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) with 50 μl of the PDGFRα-coupled protein-A agarose or protein-a agarose alone and incubated at room temperature while rotating. Protein complexes were collected by centrifugation at 500×g, washed 3 times with Tris-saline (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40) and eluted in sample loading buffer (50mM Tris-pH 6.8, 10% glycerol and 2% SDS) with 1% 2-mercaptoethanol. Precipitated proteins were separated using SDS-polyacrylamide electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were incubated in TBS containing 0.1% Tween-20 (TBST) plus 5% non-fat milk, washed, followed by incubation in TBST with antibodies specific for HA (Invitrogen) or a rabbit polyclonal sera specific for gO [59 (link)] overnight at 4°C. Membranes were washed 3 times for 10 min in TBST and incubated in TBST with horseradish peroxidase-conjugated secondary antibodies for 1h. Proteins were detected by incubating membranes in chemiluminescent reagent (Perkin Elmer) and imaged with an Imagequant LAS 4000 system (GE Healthcare).
Chin A., Liu J., Jardetzky T., Johnson D.C, & Vanarsdall A. (2022). Identification of functionally important domains of human cytomegalovirus gO that act after trimer binding to receptors. PLoS Pathogens, 18(4), e1010452.
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10

5-LOX Translocation in Differentiated HL-60 Cells

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To study the effect on 5-LOX translocation, differentiated HL-60 cells were preincubated with tested compounds or DMSO for 30 min and then stimulated by THAP or A23187 for 15 min. The nuclear and cytosol fractions were isolated by NEPER nuclear and cytoplasmic extraction reagent (Thermo Scientific). The resulting solution was heated at 95°C for 8 min. Proteins (25–50 μg) were resolved on 10% SDS-PAGE gels, transferred onto a polyvinylidene floride membrane (Millippore), and probed by a 5-LOX antibody. Membranes were exposed to chemiluminescent reagent (PerkinElmer) and visualized on a Kodak film.
Park N.Y., Im S, & Jiang Q. (2021). Different forms of vitamin E and metabolite 13’-carboxychromanols inhibit cyclooxygenase-1 and its catalyzed thromboxane in platelets, and tocotrienols and 13’-carboxychromanols are competitive inhibitors of 5-lipoxygenase. The Journal of nutritional biochemistry, 100, 108884.
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